The long term goal is to understand how retinoic acid (RA; the major biologically active metabolite of vitamin A) and bone morphogenetic protein 4 (BMP4) interact to regulate development. In general terms, RA regulates gene transcription by binding to its nuclear hormone receptors, the RARs; and BMP4 is a secreted protein that binds to cell surface receptors to initiate a signaling cascade that involves SMAD activation. Although the major BMP4 promoter in bone (1A promoter) is induced by RA, we have found that a previously described minor promoter (1B) and a novel promoter in intron 2 (i2) are repressed by PA. The i2 promoter is highly expressed in developing inner ear, whereas the 1A promoter is not. BMP4 and PA are both known to be essential for normal inner ear development. Antagonism of BMP4 inhibits development of the semicircular canals. Embryonic exposure to exogenous RA results in a similar phenotype, suggesting that PA may repress BMP4 in the inner ear, helping to restrict BMP4 expression appropriately in space and time. Indeed, repression of the novel i2 promoter by RA occurs both in an inner ear derived cell line and in vivo in mouse embryonic inner ear. Furthermore, the ability of RA to inhibit semicircular canal development is overcome by recombinant BMP4 (in chicks). This proposal seeks to address three Specific Aims: 1) Determine the mechanism by which PA down-regulates BMP4 transcription; 2) Characterize expression from the BMP4 1B and i2 promoters in space and time in developing mouse embryos; and 3) Determine the in vivo effects (in mice) of mutations that destroy the BMP4 1B and i2 promoters. The first aim is relevant to RA action in general in that the mechanism(s) of repression of target genes by PA is not well understood (in contrast to gene induction), and is of interest because PA up-regulates BMP4 in bone via a different promoter, the 1A promoter. The 2nd and 3rd Aims will establish the relative roles of the IB and i2 promoters in vivo in inner ear development, and will examine whether these promoters may play important roles outside the inner ear. Accomplishing these goals will rely on classical molecular tools to study gene regulation in cell culture and in cell free systems, as well as transgenic and gene-targeted mice to investigate the in vivo functions of the BMP4 1B and i2 promoters. The studies are relevant to understanding 1) how states of vitamin A excess or deficiency affect development; 2) molecular events that lead to normal or abnormal inner ear development; and 3) the molecular pathway of gene repression by PA.